Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce “blocking oligos”, a low-cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A. thaliana-specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots (Bromus erectus, Plantago lanceolata, Lotus corniculatus, Amaranth sp., Arabidopsis thaliana). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant-derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A. thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems-based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package.

中文翻译：

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使用简单的方法在扩增子测序中阻断宿主和非目标，深入了解微生物组多样性

分析不同的微生物组正在彻底改变我们对生物机制和生态相关问题的理解，包括元有机体（宿主 + 微生物组）组装、功能和适应。因此，多个保守的、系统发育信息丰富的基因座的扩增子测序已成为许多研究人员的工具。然而，许多系统的研究受到阻碍，因为扩增来自宿主或过多微生物的非目标 DNA 可能会丢失基本的测序深度。在这里，我们介绍了“blocking oligos”，这是一种低成本且灵活的方法，它使用标准寡核苷酸来阻止不同非目标物的扩增，并使用软件来帮助其设计。我们主要将它们应用在叶子上，在这种情况下，宿主扩增的特殊挑战盛行。一个。 在八个不同的目标基因座中应用的拟南芥特异性阻断寡核苷酸可将不需要的宿主扩增减少多达 90%。为了扩大适用范围，我们设计了通用的16S和18S rRNA基因植物阻断保守在不同的植物物种和证明他们有效地阻断从五个订单跨越单子叶和双子叶植物5种植物（为目标寡核苷酸雀 直立人，车前子 杉木，莲花 百脉，苋菜属，拟南芥 拟南芥）。这些可以增加 alpha 多样性的发现，而不会偏向 beta 多样性模式，并且不会损害植物来源的 16S rRNA 基因扩增子测序数据固有的微生物负载信息。最后，我们设计并测试了阻断寡核苷酸，以避免产孢卵菌病原体的 18S rRNA 基因扩增，证明它们在远远超出植物的应用中的有效性。使用这些工具，我们生成了对A的调查。 拟南芥叶微生物组基于针对细菌、真菌、卵菌和其他真核微生物的八个基因座，并讨论常用的扩增子测序区域的互补性，用于描述叶微生物群。通过使扩增子测序更具针对性，这种方法有可能使各种研究系统中的问题更容易处理，从而对微生物发现产生更深入的、基于系统的见解。为了在其他研究系统中快速轻松地设计用于封闭任何非目标 DNA 的寡核苷酸，我们开发了一个公开可用的 R 包。